Random peptide libraries are generally envisioned as a collection of peptide sequences in which all amino acids have been incorporated randomly into all positions of the peptide. The library will therefore contain l.sup.n different peptides where peptides of l residues have been constructed from n different amino acids. Each peptide in the mixture will have a unique sequence and all possible sequences for an l-residue peptide will be represented. Such libraries have been generated and used in various ways to screen for peptide sequences which bind effectively to target molecules and to identify such sequences.
One methodology to generate a random library involves expressing peptides of random composition on a virus such as the filamentous phage. Peptides are encoded by preparing randomized oligonucleotides (NNC/T).sub.n, where N is a random base (and T represents the stop codon). The nucleotides are inserted into the gene coding for a protein expressed on the phage surface, typically either the pVIII major coat protein or the pIII tail protein.
There are several chemical methods of preparing random libraries. One method for preparing a totally random library has been disclosed by Lam et al., Nature, 354, 82, (1991) and PCT/US91/04666 (WO 92/00091). The method involves synthesis of linear peptides on a solid support such as polystyrene or polyacrylamide resin. The resins are separated into 18 pools for incorporation of a single amino acid for each pool using standard solid phase synthesis methodology. After coupling, the pools of resin are recombined and redistributed into 18 pools again for coupling of a second amino acid. Each individual bead will contain a unique peptide sequence and, given a sufficient number of beads, all possible peptide sequences will be represented in the peptide library. The essence of the method is that the resin-linked peptide can be probed with some soluble receptor or antibody and beads which react with the probe can be isolated and individually sequenced using typical Edman sequencing chemistry. In addition, PCT/US91/08694 (WO 92/09300) discloses methodologies to provide selectively cleavable linkers between peptide and resin such that a certain amount of peptide can be liberated from the resin and assayed in soluble form while some of the peptide still remains attached to the resin, where it can be sequenced. See also, Lebl et al., Int. J. Pept. Prot. Res., 41, 201 (1993).
Another methodology disclosed by Geysen et al. J. Immunol. Meth., 259, 102, (1987) involves the synthesis of peptides on derivatized polystyrene pins which are arranged on a block in such a way that they correspond to the arrangement of wells in a 96-well microtiter plate. Individual chemical reactions can be performed on each pin in a single well and thus individual peptides can be prepared on each pin. The pins are typically probed using an Elisa type of double antibody assay which is also carried out in the microtiter wells. In the mimotope approach, random dipeptides are synthesized on the pins and probed with the target antibody. The best dipeptide is selected and a series of tripeptides based on the original dipeptide plus one more residue are prepared and probed. The best tripeptide is used as the basis of a tetrapeptide and so on until an optimized sequence up to an octapeptide has been determined. The strategy allows for incorporation of non-natural amino acids or D-amino acids into the peptides. In order to circumvent the necessity of a soluble "receptor," a cleavable linker has been described which will release the peptide from the pins involving a C-terminal .epsilon.-Lys-Pro sequence, see Bray et al., Tet. Lett., 31, 5811(1990) or via ammonolysis, see Bray et al., Tet. Lett., 32, 6163 (1991).
Another approach to de novo determination of antibody or receptor binding sequences but involving soluble peptide pools is that of Houghten et al., Nature, 354, 84 (1991). Using simultaneous solid phase synthesis, hexapeptides are prepared in which unique amino acids are coupled at two defined positions in the sequence and an equimolar mixture of amino acids is used for the couplings for the remaining four positions. The resulting peptides can be represented by the sequence O.sub.1 -O.sub.2 -X-X-X-X, [SEQ ID NO.: 1] where O.sub.1 and O.sub.2 are defined amino acids and X represents the randomized amino acids. For peptides constructed of 18 amino acids , there are 18.sup.2 or 324 different peptide mixtures or pools, each mixture consisting of 18.sup.4 or 104,976 individual peptides with a single defined residue for O.sub.1 and O.sub.2. Each of the 324 pools is assayed for activity and the best selections for O.sub.1 and O.sub.2 are used as the basis to vary the next residue, i.e., constructing peptides of the sequence A.sub.1 -A.sub.2 -O.sub.3 -X-X-X [SEQ ID NO.: 2], where A.sub.1 and A.sub.2 are the optimized residues, O.sub.3 is the defined amino acid which will be varied, and X is the random pool of amino acids. This process is repeated iteratively until an optimized sequence is obtained. The process can be streamlined considerably by preparing hexapeptides containing only one amino acid to be varied, i.e., the 18 peptides O.sub.1 -X-X-X-X-X [SEQ ID NO.: 3], the 18 peptides X-O.sub.2 -X-X-X-X [SEQ ID NO.: 4], etc. for a total of 6.times.18 or 108 peptide pools. Each pool is assayed and the best residues for each position are determined. See, Pinilla et al., Biotechniques, 13, 901 (1992) and PCT patent application WO 92/09300 (Iterex Pharmaceuticals). These techniques relate to linear peptides.
Linear peptides historically have represented relatively poor objects for drugs and for pharmaceutical design. Linear peptides are able to adopt a multitude of conformations and hence are able to bind in various ways to a single target molecule and may bind to various target molecules in various conformations. Molecules which are rigid or able to adopt a limited number of conformations bind more selectively to target molecules. In addition, linear peptides have presented difficulties in drug development and there is no rational strategy for conversion of a linear peptide into a non-peptide drug.
The use of resin-bound cyclic peptides and free cyclic peptides in combinatorial libraries are disclosed in PCT/US91/04666 (WO 92/00091) and Darlak et al., Proceedings of the 13th American Peptide Symposium, 2-193, P902 (1993). These cyclic peptides do not contain a conformationally constraining element and where cyclization is possible, these peptides generally may adopt a large number of conformations and suffer many of the same shortcomings as linear peptides.
The present invention improves the quality of the peptide and the lead for rational drug design derived from random libraries by introducing defined conformational constraints. Peptides and leads derived from the templates of this invention not only optimize side chain position but also overall conformation, which is a significant advantage in any peptide drug and any effort to rationally design non-peptide ligands. The present invention also eliminates the need for stepwise peptide sequencing, and the requirement for a soluble target molecule which are shortcomings of some of the prior random libraries.